1. Field of the Invention
The present invention relates to cell cultures and, more specifically, shape-memory polymers for use in cell culture.
2. Description of the Related Art
Living cells are remarkably complex, dynamic, and versatile systems, but the material substrates currently used to culture them are not. Although properties of the material substrate, such as surface geometry and stiffness, can direct cell lineage specification, cell-growth kinetics, cell orientation, cell migration, and cell traction, the polymeric materials commonly used in cell culture and tissue engineering only offer attached cells surfaces and structures of unchanging properties. This physical stasis of current cell culture and tissue engineering materials severely limits our ability to control cell-material interactions during cell culture and tissue engineering and, therefore, our ability to advance understanding and application of fundamental cell processes.
During tissue formation and repair, the extracellular matrix (ECM) undergoes continual biochemical turnover with corresponding architectural changes that affect structural and mechanical properties. Through cell-matrix interactions, this dynamic ECM behavior supports and regulates morphogenesis. Recent in vitro studies have begun to elucidate the principles through which this support and regulation occurs. Notably, both ECM-dependent changes of cell shape and ECM mechanical stiffness can regulate progenitor cell differentiation. As the fundamental understanding of these principles improves, there is the potential to use biomimetic scaffolds to apply these principles in regenerative medicine strategies. But the development of new scaffolds that incorporate these principles and mimic the dynamic behavior of natural ECMs has lagged behind advances in the understanding of the principles themselves. On the one hand, polymer scaffolds of controlled pore architecture provide tailored mechanical function and mass transport properties within complex 3D anatomical shapes but are fundamentally static structures. On the other hand, hydro gels can be programmed to undergo changes in structure, such as gelation following injection or controlled swelling, but sacrifice many of the benefits of a controlled architecture. Therefore, there is a need to develop advanced biomimetic scaffolds that combine the benefits of a controlled pore architecture with the ability to undergo programmed changes in architecture.
Regenerative medicine has the potential to develop therapies for previously untreatable diseases and conditions while providing technology recognized as critical to combating rising healthcare costs. The processes of tissue formation during embryogenesis and tissue repair following injury provide the blueprints for regenerative medicine. During tissue formation and repair, the architecture of the natural extracellular matrix and dynamic changes in that architecture are central to many key morphogenetic processes. Matrix architecture has profound effects on the shape of cells in the matrix, and recent studies elegantly demonstrate that cell shape regulates cell differentiation. Yet, scaffolds, the matrices of tissue engineering, currently have little or no ability to undergo programmed changes in architecture. Therefore, there is a critical need to develop advanced biomimetic scaffolds with the ability to undergo programmed changes in architecture.
Shape-memory polymers (“SMPs”) are a class of smart materials that offer mechanical action triggered by an external stimulus such as temperature change, as shown in FIG. 1, which may be useful as cell culture scaffolds. More specifically, SMPs are able to ‘remember’ one or more shapes, each determined by network elasticity, but can be stored in temporary shapes by virtue of material immobilization, commonly by vitrification or crystallization. As a simple example, a complex three-dimensional SMP shape can be compacted into a slender form (suitable for catheter delivery to the body, or to fit into an otherwise compact space) by a cycle of heating, deforming, cooling, and unloading. ‘Good’ SMPs will be those that feature elasticity during deformation and solidification (strain “fixing”) during cooling. In those cases, application of heat, light, or solvent exposure can trigger near-complete return to the equilibrium, complex shape through network chain mobilization.
Several review articles have been written on SMPs and these indicate a diversity of synthetic approaches and applications areas, the latter ranging from mechanical mechanisms to deployable space devices to surgical implements. Despite an accelerated publication frequency on SMPs exceeding 300/yr, particularly for medical applications, there are so far no reports on the utilization of SMPs in cell culture or tissue engineering.
On the other hand, numerous accounts of biodegradable SMPs have appeared that are exploited in the current application. These are: (i) polyurethanes with biodegradable soft and hard blocks; and (ii) photocured, end-linked networks with biodegradable network chains. For case (i), shape memory is possible through the elasticity of physical cross linking and strain fixing by vitrification of the soft segments. For case (ii), shape memory is possible through covalent network junctions that yield elasticity and the crystallizable network chains that can temporarily fix strains. Biodegradability and shape memory are necessary, but not sufficient, characteristics. In addition, processing into a proper form and control over cell-surface interactions are needed.
One category of materials with excellent shape-memory properties are hydro gels. Hydro gels from both natural and synthetic polymers are excellent biomaterial scaffolds for repairing and regenerating a variety of tissues and organs, such as bone, cartilage, skin, vasculature and nerves. They are attractive because: (i) they can provide a three-dimensional (3D) environment similar to the extracellular matrix (ECM), which allows diffusion of oxygen, nutrients and metabolic waste through the elastic network; and (ii) the cross-linked polymer networks are capable of absorbing water to swell, but are insoluble, to make these materials “soft” and “wet”, which improve their compatibility with biological tissues. A hydrogel will exhibit shape memory functionality if it can be stabilized in the deformed state in a temperature range that is relevant for the particular application. A typical shape memory hydrogel is a cross-linked material having a hydrophilic fraction that will swell in water and hydrophobic sections with reversible order-disorder structures controlled by temperature. While cross linking sets the permanent shape (high temperature), the ordered structure that forms at a temperature, T<Ttrans (switching transition temperature) can be used to fix secondary shapes established by deformations at a higher temperature, T>Ttrans. Heating above Ttrans triggers complete shape recovery.
Beside shape memory properties, hydro gels employed as implantable biomedical devices should also have good biocompatibility and bioactivity to facilitate cell-polymer interactions and avoid adverse physiological reactions between plants and surrounding host tissues. However, most synthetic hydro gels typically exhibit minimal or no intrinsic biological activity, which may not provide an ideal environment for culturing anchorage-dependent cells, such as endothelial cells (ECs), smooth muscle cells (SMCs), fibroblast and osteoblasts. These drawbacks prevent them from being used directly for tissue repair. Consequently, much work has been done both physically and chemically to incorporate bioactive factors and peptides into these scaffolds in order to provide them with signaling domains that have specific interactions with surrounding cells by molecular recognition. Among these functional biosignal molecules, Arg-Gly-Asp (RGD) peptide sequences derived from ECM proteins are the most widely studied cell-binding domains for the bioactive modification of scaffolds. Cells adhere to the hydro gels modified with RGD peptide sequences via particular interactions between the grafted adhesion ligands and the integrin receptors on the cell membrane.
To maintain the biological activity of the peptide upon modification, the bioactive ligand must be flexible and experience minimal steric hindrance. It has been shown that RGD sequences covalently immobilized to a hydrogel scaffold via poly(ethylene glycol) (“PEG”) spacer arms can promote cell adhesion and spreading compared with those without the PEG arms. It is hypothesized that flexible PEG arms permit biospecific receptor-ligand interactions between the peptide and cells that leads to cells behaving in response to the RGD sequences incorporated in the scaffold, while cell adhesion on the scaffold without a PEG spacer is principally nonspecific due to the sterical unavailability of the peptide incorporated in the scaffold. In addition, peptide incorporated without a PEG spacer causes more undesired protein adsorption than an equivalent amount of peptide incorporated via a PEG spacer.
Despite many recent advancements in hydrogel shape memory, there is a continued need for a biodegradable and biocompatible hydrogel based scaffold with tailored shape memory effect and good bioactivity as well as desirable mechanical property for soft-tissue applications.